Prevalence of intestinal parasitic infections and genetic differentiation of Strongyloides stercoralis among migrant workers from Myanmar, Lao PDR and Cambodia in northeastern Thailand

Intestinal parasitic infections (IPIs) remain a public-health problem worldwide, including in countries of the Lower Mekong subregion. Increases in human migration from neighboring countries might cause reemerging parasitic infections, leading to spread of parasites in the landscape. Here, we conducted a cross-sectional study to identify the prevalence of IPIs in migrant workers from Myanmar, Lao PDR, and Cambodia who were dwelling in Nakhon Ratchasima Province, northeastern Thailand. The identification of Strongyloides species and genetic differentiation of worms from migrant workers with different countries of origin was also assessed. Fresh stool samples were collected from 338 migrant workers and examined for evidence of IPIs using agar plate culture (APC) and the formalin-ethyl acetate concentration technique (FECT). Among those nine samples positive for nematodes by APC, the Strongyloides or hookworm species present was confirmed using the polymerase chain reaction (PCR) followed by DNA sequencing. This revealed eight cases of Strongyloides stercoralis infection and one of Necator americanus. Fifty-one out of 338 individuals (15.09%) were positive for IPIs using FECT and APC. Eggs of Opisthorchis-like flukes were the most common parasite (11.83% of samples), followed by S. stercoralis (2.37%), Entamoeba coli (1.50%), hookworm (0.89%), Taenia sp. (0.60%) and Hymenolepis nana (0.30%). The genetic differentiation of S. stercoralis recovered from migrant workers with different countries of origin was analyzed. Specimens of S. stercoralis isolated from workers from Lao PDR, Cambodia and Myanmar were genetically similar to those sequenced from Thailand. However, there were population-genetic differences between S. stercoralis from these Southeast Asian countries and other regions of the world. This study demonstrated that IPIs were prevalent in migrant workers in the northeastern region of Thailand. Our findings provided molecular confirmation of the presence of S. stercoralis and explored the genetic differentiation of S. stercoralis from those infected migrant workers. An effective anti-parasitic drug should be provided for migrant workers and its administration enforced.


Introduction
Intestinal parasitic infections (IPIs) caused by protozoan and helminth parasites remain a public-health problem worldwide [1][2][3], including in countries of the Lower Mekong subregion [4][5][6]. The world's population is increasingly mobile, with many travelling from less-developed to more-developed countries for employment opportunities. Thailand is a middle-income country in which construction, food services, manufacturing, and agricultural sectors have all seen economic expansion. Employment in these sectors often attracts migrant workers. In Thailand, most such workers come from Cambodia, Lao PDR, Myanmar and Vietnam [7]. These countries have all reported a high prevalence of IPIs [8][9][10]. Infected migrants arriving from these countries may cause reemergence of parasitic infections with the potential to spread across the country. To prevent this, control strategies are urgently required.
In Thailand, the Ministry of Public Health has a health-screening policy with which migrant workers must comply before applying for work. This policy also mandates compulsory administration of 400 mg per kg body weight of albendazole to control IPIs [11] although, as will be seen, this is rarely enforced in practice. Recent studies have reported IPIs in migrant workers in Thailand detected using parasitological methods [12][13][14]. However, these techniques require an expert microscopist and are inadequate to distinguish between certain parasite species, especially Strongyloides spp. [15,16]. It is necessary to use molecular approaches to confirm the identity of such parasite species [17,18].
Nematodes of the genus Strongyloides are soil-transmitted helminths (STH) which cause strongyloidiasis [19,20]. The two medically important species of Strongyloides are S. stercoralis and S. fuelleborni [21][22][23][24]. Molecular identification of Strongyloides spp. is largely based on DNA sequences of mitochondrial (cytochrome C oxidase subunit 1 (cox1)) and nuclear ribosomal RNA regions (18S rDNA) [25,26]. Infection with S. stercoralis is more common than with S. fuelleborni and can have serious consequences, causing death in immunocompromised patients, those infected with human T-lymphotropic virus 1 (HTLV-1), organ-transplant patients, and those undergoing corticosteroid therapy [27]. In such cases, the parasites can disseminate to various organs of their hosts [28].
Although previous studies using the cox1 gene have identified S. stercoralis in Thailand by PCR and DNA sequencing [22], migrant workers living in Thailand have not been investigated using this approach. It is possible that molecular studies on the parasites carried by these workers will demonstrate genetic differences related to their country of origin. Understanding the prevalence of IPIs in migrant workers and the extent of genetic differentiation from different geographical origins will be useful for health policies aiming to reduce the transmission of parasites across the country and also to increase understanding of the levels of regional genetic variation [29].
Herein, we aimed to identify IPIs in migrant workers in Nakhon Ratchasima Province, Thailand. We also used cox1 DNA and 18S rRNA sequences to confirm the presence of S. stercoralis in migrant workers and to evaluate genetic variation in this parasite associated with their country of origin and compared with global geographic differences.

Human ethics statement
This study was approved by the human ethics committees of Mahasarakham University (Human Ethics number: 063-354/2021). Permission was granted by the owners of several factories (eg. food, plastic production, and tapioca starch factories), and by migrants working there who were willing to participate in the current study. All workers recruited to the study completed and signed the written informed-consent forms. These forms had been translated into the language of each migrant worker's home country. At the end of the study, all individuals infected with S. stercoralis were treated with a single dose of ivermectin at 20 μg/kg body weight, while other IPIs detected were treated with appropriate anthelminthic drugs.

Population and sample size calculation
This cross-sectional study was conducted between March 2021 and January 2022 involving four factories in four districts (Muang, Sung Noen, Khon Buri, and Pak Chong Districts) in Nakhon Ratchasima Province, Thailand. This province was chosen because it is one of the most industrialized in northeastern Thailand. A total of 7351 legal migrant workers were living in the province from October 2020 to July 2021, as reported by the migration division of Thailand. These included 4845 migrants from Myanmar, 2010 from Cambodia, and 496 from Lao PDR [7].
The required sample size was calculated using an estimated population proportion formula [30] based on the prevalence of S. stercoralis infection at 31.1%, as previously described in Cambodia in year 2017 [31] with 95% confidence interval (z = 1.96), power at 80% (β = 0.20). The required sample size was calculated as 315 individuals. However, a total of 338 participants had returned the informed consent form, so we included all individuals in the study.

Stool sample collection
Migrant workers were briefed about the objectives of this study by our trained translators before stool samples were collected. After the consent form was obtained and the questionnaire was completed, stool containers were provided to the participants. The process of collecting the stool samples was also explained by our trained translators. Fresh stool samples were collected on the next day in the morning. Agar plate culture (APC) was used to detect S. stercoralis and/or hookworm infection [32], and the formalin-ethyl acetate concentration technique (FECT) was used to diagnose other intestinal parasitic infections (protozoans and helminths). Approximately 2 g of fresh stool sample were used for FECT and the protocol was performed in duplicate for each sample as previously described [33]. For the APC method, a total of 4 g of stool sample was plated on the center of an agar culture plate immediately after collection. The agar plates were then sealed with paraffin film and carried to the laboratory at Mahasarakham University. The plates were incubated at room temperature for 4-5 days and examined daily using stereo microscopy. Worms were collected from positive plates. The surface of positive agar plates was washed with 70% alcohol and the washings sedimented by centrifugation at 1500 rpm for 5 min. Some worms were initially examined for their morphological structure under light microscopy after staining with 1% iodine in potassium iodide solution. The remaining worms were kept at -20˚C for DNA extraction and confirmation of parasite species using DNA sequence data [34]. used in Myanmar, Cambodia and Lao PDR. Participants completed the questionnaires by themselves.

DNA extraction, amplification and sequencing
Two worms were taken from each positive culture, individually crushed with a disposable polypropylene pestle (Bellco Glass, Vineland, NJ, USA) and DNA extracted using the Nucleospin Tissue kit (Macherey-Nagel GmbH & Co, Duren, Germany). The protocol was performed according to the manufacturer's instruction. The quality of genomic DNA was checked using a Nanodrop (NanoDrop Technologies, Wilmington, DE, USA). Conventional PCR was performed to amplify an 835-bp region of the mitochondrial cytochrome C oxidase subunit 1 (cox1) gene and nucleotides between positions 843 and 1410 (GenBank accession numbers: AF279916) of the 18S rRNA (hyper-variable region IV (HVR-IV)) gene of S. stercoralis. When cox1 and 18S rRNA amplification were negative, a 696-bp portion of the ITS1 and ITS2 regions of the nuclear ribosomal operon was amplified for hookworm detection and identification. PCR conditions and specific primers for S. stercoralis and hookworm were used as previously described [34][35][36]. The PCR products were sequenced using the Applied Biosystems 3730 × I DNA Analyzer and ABI Big Dye Version 3.1 (Applied Biosystems, Foster City, CA), using PCR primers as sequencing primers. All mitochondrial cox1, 18S rRNA and the ITS1 and ITS2 regions of the ribosomal DNA sequences were submitted to the GenBank database and used in BLASTn searches (http://www.ncbi.nlm.nih.gov/).

Construction of phylogenetic trees
DNA sequences from the mitochondrial cox1 and 18S rRNA and the ITS1 and ITS2 regions of the ribosomal DNA genes were aligned and trimmed using the BioEdit sequence alignment editor program [37]. The phylogenetic trees were constructed using the maximum likelihood method (ML) with 1000 bootstrap re-samplings in the MEGA-X software [38]. The most appropriate substitution model selected was the Hasegawa-Kishino-Yano model [39]. Publicly available sequences of S. stercoralis, S. fuelleborni, S. ratti and hookworms (Necator americanus and Ancylostoma duodenale) from humans and dogs in different countries were used for reference.

Genetic differentiation of S. stercoralis from migrants from different countries
The pairwise number of differences between cox1 sequences was used to explore populationgenetic differentiation of S. stercoralis by country of origin of migrant workers (Myanmar, Lao PDR and Cambodia). Published sequences in GenBank from Thailand (n = 58), Myanmar (n = 88), Lao PDR (n = 37) and Cambodia (n = 7) were included. Also included were sequences from China, Japan, Iran, African and South American continents (S1 File). The GenBank database was searched by name of parasite, gene, sequence coverage and country and representative sequences were selected randomly. Population-pairwise genetic differentiation (F ST ) was determined in Arlequin 3.5.2 software [40]. The standard F ST calculations were tested using 1000 permutations based on the Tajima-Nei model of substitution. An F ST value greater than 0.15 and p-value less than 0.05 can be considered to indicate significant differentiation between populations [41].

Statistical analyses
Demographics and socioeconomic status of participants were described as numbers and percentages of individuals. Continuous data were described as mean and standard deviation (SD).
Infection with parasites was reported as frequency and percentage. These analyses were conducted using STATA software version 10.1 (Stata Corporation, College Station, TX, USA).

Study population and characteristics
We analyzed data from 338 migrant workers who completed and signed the informed-consent forms, completed the questionnaire and provided stool specimens. The participants included 151 migrant workers from Myanmar, 128 from Cambodia, and 59 from Lao PDR. The migrants comprised 184 males (54.44%) and 154 females (45.56%). The average age of participants was 31.76±8.21 years, and the average number of years of living in Thailand was for 6.94 ±4.05 years. Most (87.28%) resided in workers' dormitories and almost none of the workers had ever been treated for helminths (98.22%) ( Table 1).
Overall, the prevalence of IPIs according to FECT and APC methods was 15.09% (51/338) as shown in Table 2. Opisthorchis-like eggs were the most common infections (11.83%), followed by S. stercoralis (2.37%), Entamoeba coli (1.50%), hookworm (0.89%), Taenia sp (0.60%), and Hymenolepis nana (0.30%). Using the FECT alone, 306 samples were examined and 43 individuals (14.05%) were found to be infected with IPIs. Of the 338 stool samples examined using the APC method, nine (9/338; 2.66%) were positive for nematode worms only (3 cases from Lao PDR; 2 cases from Myanmar; and 4 cases from Cambodia) ( Table 2). Based on morphological examination of the nematodes, eight samples were identified as Strongyloides spp. and one sample was identified as hookworm. Most participants had not undergone stool examination for parasitological diagnosis in the previous year or received any medication for intestinal parasitic infections during their time in Thailand (Table 1 & S1 Table).

Molecular confirmation
The identity of the nematode parasites found in nine samples by APC was confirmed using DNA sequence data. One nuclear ribosomal sequence was identified as representing N. americanus ( Fig 1A) and eight cox1 ( Fig 1B) and 18S rRNA sequences were identified as belonging to S. stercoralis (S1 Fig). Our cox1 sequences from migrant workers all fell within the type A clade of Nagayasu et al. (2017) [42], in which both human and dog S. stercoralis isolates were located. The corresponding GenBank accession numbers are ON954817-ON954824 for S. stercoralis cox1 and ON954801-ON954808 for 18S rRNA sequences, while OP002314 is the accession number for the ITS1 and ITS2 regions of the ribosomal DNA region of N. americanus.

Genetic differentiation of S. stercoralis
For this analysis, a "population" was regarded as including all cox1 sequences from a single country (or region, China, Japan, Iran, African and South American continents). For some analyses, sequences from migrant workers in Thailand were assigned to further "populations" according to their country of origin. The pairwise F ST values indicated varying degrees of genetic differentiation of S. stercoralis between nationalities sampled in this study. No sequences of S. stercoralis from migrant workers from Lao PDR (3 sequences), Cambodia (3 sequences) and Myanmar (2 sequences) workers were significantly different from those of S. stercoralis in Thailand (58 sequences) (Fig 2A). In contrast, S. stercoralis sequences (n = 2) from Myanmar migrant workers who lived in Thailand were genetically different from worms from their own country of origin, but not significantly so. When cox1 sequences of S. stercoralis from Thailand, Lao PDR, Cambodia, and Myanmar were combined into a single "population" (Southeast Asia), this population was statistically significantly different from those from other regions (China, Japan, Iran, African and South American continents) (Fig 2B).

Discussion
In this study, we explored the prevalence of IPIs in migrant workers from Myanmar, Lao PDR and Cambodia who resided in Nakhon Ratchasima Province, northeastern Thailand. We used

PLOS ONE
Intestinal parasitic infections and genetic differentiation of S.stercoralis among migrant workers in Thailand molecular evidence (mitochondrial cox1 gene and 18S rRNA genes) to confirm the identity of S. stercoralis and to explore genetic differentiation between samples from workers from different countries. Fifty-one out of 338 individuals (15.09%) were positive for IPIs using FECT and APC methods. The most common parasitic infection was due to Opisthorchis-like eggs, followed by S. stercoralis, E. coli, hookworm, Taenia sp., and H. nana, in that order. These findings illustrate that IPIs remain prevalent in the Greater Mekong subregion. The finding of Opisthorchis-like eggs is of concern. This is a food-borne helminth that is a major risk factor for cholangiocarcinoma in those chronically infected [43]. When using only the APC, nine of 338 participants were positive for nematode larvae. Sequence analysis confirmed that eight of the samples sequenced for cox1 and 18S rRNA genes belonged to S. stercoralis, while a nuclear ribosomal sequence (ITS1 to ITS2) identified the remaining sample as N. americanus. Application of such molecular techniques is an important way to distinguish S. stercoralis species and hookworm infections in areas where they overlap. The overall prevalence of IPIs (as detected by FECT) of 14.05% was lower than reported in previous studies of migrant workers in the same area of Thailand, which found rates of infection to be 24.07%

PLOS ONE
In this study, we also assessed the genetic differentiation of S. stercoralis samples from infected migrant workers from different countries. Such analysis will shed light on the parasite's origin and spread [50]. Our results from the pairwise F ST values found that sequences of worms isolated from migrants from Lao PDR, Cambodia and Myanmar were similar to those from Thailand, indicating close genetic relationship and high gene flow. This is consistent with a previous report, based on 18S rDNA and cox1 sequences, that S. stercoralis populations were not very differentiated between countries within the Southeast Asian peninsula [26]. Parasite gene flow is typically strongly influenced by host migration [29]. Given the long span of time through which people have moved throughout Southeast Asia [51,52], such genetic mixing might be expected. When we extended the analysis to include cox1 sequence data from other parts of the world, we found significant differentiation between Southeast Asia (Thailand, Lao PDR, Cambodia and Myanmar) and the other regions (China, Japan, Iran, African and South American continents), supporting geographic restriction of gene flow.
Several parasites, including S. stercoralis and O. viverrini, can persist in their host for many years. Almost none of the migrant workers had actually been given anthelmintic treatment when (or before) they arrived (Table 1). If they have brought these parasites from their home countries, then the IPI-infected migrant workers found in our study may act as carriers and distributors of parasites which may interrupt the success of IPI control programs in Thailand.

PLOS ONE
Screening programs, including diagnostic screening and provision of an effective drug treatment, such as ivermectin (for S. stercoralis) and praziquantel (for intestinal and liver flukes and tape worms) to alien workers are important for management of IPIs. This will benefit both the government and employers to improve the general health of their workers that will further translate to better productivity and will also reduce the transmission of parasites across the country [53].
The limitations of this study are (a) the small proportion of migrant workers infected with IPIs, (b) stool samples were collected only once from each participant and the quantity was sometimes insufficient for both APC and FECT (thus our figures are likely an underestimate of the prevalence of these parasitic infections) and (c) we cannot be certain whether the infections detected had been acquired in their home countries or after arrival in Thailand. Either scenario is possible given that S. stercoralis infections can be long-lived, that almost none of the workers had actually been treated for worms and that there was little or no genetic distinction among the different source and host countries. Furthermore, for genetic analysis of S. stercoralis, sequences from migrant workers were too few for robust analysis. In future work, larger samples sizes from migrant workers will be of value. Stool samples should be collected over multiple consecutive days from a greater number of individuals.

Conclusion
In this work, IPIs were found to be prevalent in migrant workers in the northeastern region of Thailand. The prevalence of S. stercoralis infection in migrant workers was low in comparison with the local Thai community. We confirmed the presence of S. stercoralis in eight workers and of N. americanus in one worker using a molecular approach. Sequences from a portion of the cox1 gene were used to explore the levels of genetic differentiation of S. stercoralis from migrant workers in Thailand with data from their country of origin. There was significant genetic differentiation between sequences from Southeast Asia and those from other parts of the world but little or no differentiation among the countries represented from Southeast Asia.